1. Field of the Invention
The present invention relates to a technique for efficient isolation of long nucleic acids and short nucleic acids from a sample containing long nucleic acids and short nucleic acids via safe and convenient operations. In addition, the present invention can be utilized as a technique for isolation of genomic DNA and total RNA (e.g., messenger RNA, ribosomal RNA, and transfer RNA) or of genomic DNA and plasmid DNA.
2. Background Art
Nucleic acids are related to genetic information in organisms, and exist in various forms such as genomic DNA, plasmid DNA, messenger RNA, ribosomal RNA, and transfer RNA, which are functionally different from one another.
Based on analysis of these nucleic acids, very important molecular biological information can be obtained. Upon analysis of various types of nucleic acids, in general, it is preferable to carry out pretreatment comprising isolation of nucleic acids of interest from a biological sample containing different types of nucleic acids. For instance, upon messenger RNA analysis for the purpose of gene expression analysis, total RNA is isolated from genomic DNA, which can act as an inhibitor during messenger RNA analysis.
In general, phenol/chloroform extraction has been known as a method for total RNA isolation from genomic DNA in a biological sample (Analytical Biochemistry, 162, 156-159 (1989)). This method comprises: (1) dissolving a biological sample in a guanidine thiocyanate solution and adding an acidic buffer solution, a phenol solution, and a chloroform solution thereto in that order, followed by mixing; (2) separating the mixture into an aqueous phase containing RNA, and an interphase and an organic solvent phase containing insolubilized DNA and denatured proteins via centrifugation; (3) adding ethanol or isopropanol to the aqueous phase containing RNA; and (4) allowing insolubilized RNA to selectively precipitate via centrifugation. Compared with conventional ultracentrifugation methods, this method is more efficient in terms of RNA isolation; however, highly hazardous phenol and chloroform must be used in the method, which is problematic.
As examples of a method for nucleic acid isolation that requires no use of phenol, chloroform, or the like, and requires no operations such as ethanol precipitation or isopropanol precipitation, methods utilizing nucleic acid binding properties with reference to a solid phase containing silica in the presence of a chaotropic agent have been known (B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. USA, 76 (2), 615-619 (1979), R. Boom et al., J. Clin. Microbiol. 28 (3), 495-503 (1990)). Also, methods for DNA and RNA isolation to which the latter method is applied have been reported (JP Patent Publication (Kokai) No. 2004-340839 A, No. 2002-187897 A, No. 2000-505295 A, No. 2002-534080 A, and No. 2004-201607 A). However, such methods result in insufficient DNA and RNA isolation, so that isolated RNA contains a predetermined amount of DNA.